1

CHAPTER- I

BIOACTIVE METABOLITES OF MARINE ORGANISMS

1

BIOACTIVE METABOLITES OF MARINE ORGANISMS

Terrestrial plants have been used to treat human diseases since the ancient ages.

Studies of the secondary metabolites of terrestrial plants were begun in the 1800’s1and

have proven to be rich sources of natural drugs that are used for the treatment of fatal

diseases such as cancer (taxol) and microbial infections (penicillin). However, due to

resurgence of pathogenic microorganisms and parasites that have developed resistance to

traditional chemotherapies, natural products chemists are increasingly turning to new

sources in the search for biologically active compounds, and specifically to marine

realm.2,3

Life on earth arose from the ocean, and living marine resources continue to

provide essential ecosystem services on which all life depends. Only in recent years has

the extent to which the Ocean is a host to a vast diversity of species and ecosystems been

recognized.

Only in the last 30 years have the seas begun to yield the secrets of the deep ocean

floor. For example, the deep ocean is home to communities of organisms whose

productivity is based on chemosynthesis instead of photosynthesis, the latter being the

process by which most plant life on the earth and in the sea converts sunlight into usable

biological energy. Other whole new ecosystems have been discovered in the ocean in

recent years, and the vast majority of species remain to be discovered.

The seas and oceans are estimated to be 141 million square miles in area and

cover three-fourth’s of the earth’s surface and harbours over 500 million species such as

algae, soft corals, sponges, gorgonians etc. in about thirty phyla 4 (Fig. I-01).

1

2

It takes a lot of energy to produce a natural product, so why do the organisms

bother? The answer is that most of the organisms are sessile as adults, meaning that they

grow attached to something else, perhaps a rock or another animal. As larvae, most of the

invertebrates can get around but once they enter their juvenile phase, they select a home,

settle there and metamorphose into adulthood if they choose any advantageous spot. Now

as they are sessile they have to keep other animals from moving into their space and

should protect themselves from temperature ranges from below freezing temperatures in

Antarctic waters to about 350 0C in deep hydrothermal vents and pressure range (1-1000

atm) which can turn a man into a jelly fish. !

This extensive variability has equipped many marine organisms with the

appropriate mechanisms to survive in a hostile milieu in terms of extreme temperatures,

changes in salinity and pressure, as well as overcoming the effects of mutation, bacteria

and viral pathogens. Marine organisms especially those which are sedentary in nature

have evolved biochemical and physiological mechanisms that include the production of

bioactive compounds for such purposes as communication, protection against predation,

infection and competition. Because of the physical and chemical conditions in the marine

environment almost every class of marine organism exhibits a variety of molecules with

unique structural features.

Difference in physical and chemical environment on the land and seas naturally

causes substantial variation in the nature of metabolites and their biogenesis. Halogens

and isocyanide groups appear frequently as substituents in the metabolites of algae or

sponges or coelenterates; yet these functionalities are very rarely observed as products of

3

terrestrial biochemistry. The metabolites of marine origin often differ in absolute

configuration (or stereochemistry) from their terrestrial counter parts. It is, however,

pertinent to note that the marine environment provides different biosynthetic conditions

to those found on land. 5 By the buffering action of sodium carbonate and bicarbonate

the pH of sea water is always maintained at 8.2-8.5. Sea water contains up to 40% salt

and has an osmotic pressure 15-25 atm.6 In order to cope with such an environment the

marine organisms may have specialized cell structures, especially the composition of the

membrane.

The composition of marine secondary metabolites, while in part, identical to those

from terrestrial plants and animals, is also significantly modified by other factors such as

the aquatic medium, partial availability of sun light up to certain depth and easy mobility

of the nutrients both organic and inorganic, besides the absence of human predation. The

halide rich sea water environment, consisting of Cl-, 19,000 mg/liter; Br-, 65 mg/liter and

I-/IO3-, 5 X10-4 mg/liter, has readily allowed marine organisms to incorporate bromine,

chlorine, and iodine in that order into covalent organic structures. Furthermore, these

elements are not only incorporated into diverse structural types, but they appear to play

essential role in terpene biosynthesis. The utilization of halogens in terrestrial secondary

metabolism is, by comparison, a rare process observed in only a few microorganisms.7

The halogenation process attains a major significance in the sea.

The organic compounds obtained by solvent extraction of a marine organism are

considered to be the secondary metabolites of that organism. Secondary metabolites are

derived from the primary metabolites (amino acids, proteins, fats and polysaccharides) of

the organism. Primary metabolites are needed for the basic cell metabolic processes but

4

the secondary metabolites are not needed in the cell metabolism. The exact function of

the secondary metabolites is not clear. Probably they are the chemicals produced by the

organism in response to environment stresses and also to serve as defense agents against

predators.8 The sea is a potential source of bioactive compounds. The bioactive

compounds could be compounds, which are directly useful in human medicine or as

allelochemics,9 i.e. as reagents by which organisms of one species affect the growth,

health, behaviour or population biology of another species (excluding substances used

only as food by the second species). Examples of the later type are found abundantly in

marine natural products. It is not surprising because to survive in the competitive marine

environment, the marine organism has to produce agents to meet different requirements,

for example, to drive away its predators, to maintain and expand its living space and for

its successful reproduction. As a result of these requirements several bioactive

compounds are possible from the numerous kinds of predator-prey combinations

occurring in the marine environment.

Metabolites of marine organisms are often believed to play an active role in

chemical defense.10,11 The metabolite which is responsible for the chemical defense

mechanism may be present during the active growth of the marine organisms or of

reproduction of the organism alone (seasonal variation), in which case a role as feeding

deterrents or as protectors of new tissue or offspring may be implied, where a more

general defense role is inferred the chemical is likely to be present at all times. Changes

in metabolic composition in sponges in response to environmental changes have been

detected.12 In many cases the compounds that are produced in artificial environments

(such as under laboratory cultures) are identical to those produced in situ. 13 In total

5

contrast, seemingly identical colonies of organisms such as sponges or algae or soft

corals that have been collected in the same location may sometimes have different

chemical composition.

Due to technical barriers there has been a lack of extensive marine folk medicine

in the western world. A Chinese pharmacopoeia recommends seaweed based recipes for a

number of disorders such as pain, abscesses, menstrual disorders and cancer. Seaweed

remedies were also used by the San blas Indians in Panama.14 The relationship between

marine sponges and medicines goes back to Alexandrian Physicians and was described

by Roman historian Plinius. Physicians used sponges that were saturated with iodine to

stimulate coagulation of the blood. Sponges were soaked with pure wine and put on the

left part of the chest in case of heartaches and soaked in urine to treat bites of poisonous

animals. They were used against all kinds of wounds, bone fractures, dropsy, stomach

aches, infectious diseases, etc. At least since the 18th century, Russian, Ukrainian and

Polish physicians have used a fresh water sponge called Badiaga for the treatment of

patients.

The dry powder of this sponge is rubbed on the chest or back of patients with lung

diseases or on the sore places in cases of foot and leg aches such as rheumatism.15

Oficjalski (1937) discovered that Badiaga is not really one sponge, but mixtures of

several freshwater sponges that differ depending on the region. In Poland it consisted of

powder of “Euspongilla lacustris, Ephydatia fluviatilis and Meyenia muelleri,” while the

Russian Badiaga was a mixture of “Euspongilla lacustris, Ephydatia fluviatilis, Spongilla

fragilis and Carterius stepanowi.” He suggested that the high iodine concentration in all

sponge species gives rise to the wholesome effect of Badiaga. At present stodal, syrup

6

containing roasted ‘Spongia officinalis’ is used for homeopathic treatment of dry and

asthmatic cough in the western world.16

The deep knowledge about nerve transmission has been learnt using squid and its

giant nerve axons and the mesenteries of vision have been unraveled using the eyes of

horse shoe crabs, sharks and skates. The surf clam is proving an excellent model for the

cell cycle and its regulation while the sea urchin is a model for understanding the

molecular basis of cellular reproduction and development.17

By the early 1950s, researchers began to view the oceans as a new and untouched

source of potentially useful compounds. Several research groups around the world have

joined hands in marine chemical research and the author has made an attempt to present

their contributions in a nutshell in the following pages. The activities of these metabolites

are immense and mainly depend on the ingenuity of man (Chart I-01).

In recent years, marine natural product bioprospecting has yielded a considerable

number of drug candidates. Most of these molecules are still in preclinical or early

clinical development (Table I-02) but some are already on the market, such as

cephalosporins, cytarabine (Ara-C), vidarabine (Ara-A), ecteinascidin 743 (ET 743) and

ziconotide.

The unusual nucleosides spongouridine and spongothymidine, isolated from

marine sponge Tethya crypta in 1950s, served as lead structures for the development of

commercially viable anti-viral drug Ara-A and the anticancer drug, Ara-C, while

Cephalosporin C, isolated from marine fungus Cephalosporium acremonium in 1940s,

led to the development of the cephalosporin antibiotics. But serious attempts to tap the

vast potential of marine organisms as sources of bioactive metabolites were started only

7

in late 1960s by the interdisciplinary marine research groups at the Scripps Institute of

Oceanography in California, the University of Hawaii and Bedford Institute of

Oceanography, Canada.

The discovery of sizeable quantities of prostaglandins, which had just been

discovered as important mediators involved in inflammatory diseases, fever and pain,

isolated from the gorgonian Plexuara homomalla by Weinheimer and Spraggins18 was

usually considered as the “take-off point” of any serious search for drugs from the sea.

In the early 1970’s researchers at the University of Oklahoma screened about 2000

extracts of diverse marine species and found that nine percent contained compounds with

anti-tumor activity. Inspired by these results and other screening studies, Hoffmann-

LaRoche of Switzerland entered the field in a big way by establishing the Roche

Research Institute of Marine Pharmacology near Sydney, Australia. Even after six years

of intensive research, not a single drug could be developed and understandably the

company closed its venture in 1980. During the 1990’s, workers at several laboratories,

including those of Dr.Richard Moore (University of Hawaii), Dr.William Gerwick

(Oregon State University) and colleagues, had begun to screen extracts of cyanobacteria

(blue-green algae) for various biological activities using predominantly mechanism and

enzyme based assays.

However, search for pharmaceuticals from marine resources continued both in

United States and Japan. Major drug makers, Upjohn, Novartis, Johnson and Johnson,

Knoll, Eli Lilly and others, had started supporting marine natural product research in

University Laboratories worldwide. Pharma Mar, a Spanish company dedicated wholly

to development of anticancer drugs from marine organisms was started in 1986. It has a

8

collection of about 40,000 marine organisms, which has yielded about 150 anticancer

compounds, of which 14 are preclinical candidates, five are in late-stage evaluation and

four are in clinical development. Aplidine, a cyclodepsipeptide from a tunicate has been

approved in Europe for acute lymphocyte leukemia and has already received marketing

authorization. Mera Pharmaceuticals exploring microalgae and Cerylid (Victoria,

Australia) with a good collection of marine invertebrates are the other companies actively

pursuing the development of drugs from marine natural products.

It is now understood that the search for drugs from the ocean resources is rather

slow and arduous task and many laboratories around the world have continued programs

to achieve the goal. Beginning the studies on a humble number of compounds from

marine sources, copious literature on marine natural products and their utility has

emerged. In an article by Grant and Mackie “Drugs from the Sea-Fact or Fantasy?” it is

concluded that marine organisms are potential resources of bioactive compounds.19 The

chemical and biological perspectives have been reviewed by experts in the field of

marine natural products in “Marine Natural Products: Chemical and Biological

Perspectives” edited during 1978-83 by Professor P.J.Scheuer of the University of

Hawaii.20 In 1987, he initiated a new series of volumes “Bioorganic Marine Chemistry.”21

Halstead’s22 three volume series “Poisonous and Venomous Marine Animals of the

World” covers the history, biology, morphology, toxicology, pharmacology and

chemistry of all known poisonous and venomous marine organisms. Marderosian 23 in his

inspiring review article “Marine Pharmaceuticals” alerts the pharmaceutical community

about the enormous potential of the sea as a storehouse of new and different

pharmaceuticals of many types. Kaul and Daftari 24 in their article “Marine

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Pharmacology: Bioactive Molecules from the Sea” focused on pharmacological activities

of pure compounds isolated from marine organisms. Garson 25 reviewed the biosynthesis

of marine natural products in an excellent report “Biosynthetic studies on Marine Natural

Products”. Riguera 26 detailed interesting bioactive compounds from marine organisms

and Kornprobst et al. 27 reviewed sulfated compounds from marine organisms. Bakus 28

expounded the chemical ecology of marine organisms in an informative and inspiring

report “Chemical Ecology of Marine Organisms: an overview”. Colwell 29 illustrated the

immense potential of biotechnology to the development of marine sciences in an

inspiring article “Biotechnology in Marine Sciences”. Burja et al.30 reviewed various

compounds isolated from marine cyanobacteria and their biological activities in an article

“Marine Cyanobacteria- a prolific source of natural products”. Biotechnological

applications of cultivation of marine sponges were discussed by Osinga et al.31 in an

article ‘Cultivation of marine sponges for metabolite production: applications for

biotechnology?’ Forty-two articles on various aspects of marine biotechnology were

featured in a special issue of the Journal of Biotechnology.32 Berman et al.33 reviewed

‘Marine microorganisms as a source of new natural products’ while Pietra 34 discussed

prospects of secondary metabolites from marine microorganisms in the article ‘Secondary

metabolites from marine microorganisms-bacteria, protozoa, algae and fungi-

achievements and prospects’.

The individual reviews in the edition of “Chemical Reviews: Marine Natural

products Chemistry”35 which include chemical studies of marine bacteria, the

biosynthesis of marine natural products, bioactive peptides and amino acid derived

metabolites from the marine organisms, marine toxins and alkaloids etc. Kelecom 36

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reviewed ‘Chemistry of marine natural products: yesterday, today and tomorrow’ while

Scheuer37 presented an introductory educational article ‘Products of chemistry -

Exploring the Ocean-Stating the case for the Chemistry’. A book entitled Drugs from the

Sea consisted of a number of reviews running the whole gamut of marine drug discovery

38-47, like ‘Marine microorganisms and drug discovery: current status and future

potential’, ‘Microalgae as a drug source’, ‘Search for biologically active substances from

marine sponges’, ‘Cytotoxic substances from opisthobranch molluscs’, ‘-Conotoxin

MVIIIA: from marine snail venom to analgesic drug’, KRN-7000 as a new type of

antitumour and immunostimulatory drug’, ‘Zoanthamines, antiosteoporotic alkaloids’,

‘Symbiotic bacteria in sponges: sources of bioactive substances’, and ‘The halichondrins:

chemistry, biology, supply and delivery’. Cimino and Ghiselin 48 gave an evolutionary

narrative of marine natural products chemistry which included a taxonomic survey while

Capon 49 highlighted the molecular diversity seen in the results obtained by the

University of Melbourne’s marine natural products group in an article ‘Marine

bioprospecting – trawling for treasure and pleasure’. A database, MarinLit, dedicated to

marine literature is maintained by the marine chemistry group at University of

Canterbury, New Zealand.50 The task of tracking and cataloging the steady stream of

fascinating new structures has been undertaken up to 2002 by D. John Faulkner51 and

later by research group at University of Canterbury52 in periodical reviews, “Marine

Natural Products”. In addition, numerous books53-55, symposia proceedings56-58,

reviews59, specialist and non-specialist articles 60-67 all devoted to marine natural products

have been published. In the year 2003 the members of the Marine Drugs and Food

11

Institute at Ocean University of China in Quindao, China launched a journal “Marine

Drugs” dedicated to marine chemistry and pharmacology. 68

Schwartsmann69 discussed semisynthetic and biocatalytic methods for

enhancement of structural diversity and bioactivity along with latest developments of

underwater breathing apparatus in an article ‘Enhancement of marine natural product

structural diversity and bioactivity through semisynthesis and biocatalysis’. Newman et

al. 70 summarized preclinical and clinical trial data for a range of marine natural products

in an article ‘Marine Natural Products and Related Compounds in Clinical and Advanced

Preclinical Trials’. There has been a steady and continuous flow of research papers on

marine natural products in almost all scientific journals. Marine organisms biosynthesize

compounds as simple as iodoform and as complex as maitotoxin.71,72 The number of

compounds that have been isolated from various marine organisms has virtually soared

and now exceeds 17,00043,73 with hundreds of new compounds still being discovered

every year.25,26,49-53 From 1969-2008 approximately 400 patents on bioactive marine

natural products were issued. The marine derived products currently on the market are

listed in Table I-01

Although the number of clinically used drugs developed from marine sources is

less, a number of promising compounds that have been identified are either already at

advanced stages of clinical trials (Table I-02) or have been selected as promising

candidates for extended preclinical evaluation with the combined efforts of natural

product chemists and pharmacologists. The stronghold of marine natural products that

entered clinical trials is in the area of cancer treatment. A brief account of these is given

below.

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Table I-01 Marine derived drugs in market

Product Application Original source Method of production

Ara-A Antiviral drug Marine sponge Microbial fermentation of analog

Ara-C Anticancer drug Marine sponge Chemical synthesis of analog

Okadaic acid Molecular probe: phosphatase inhibitor Dinoflagellate Cell culture

Manoalide Molecular probe: phospholipase A2 inhibitor

Marine sponge, Luffartella vartabilis

Wild harvest of sponge

Vent TM DNA polymerase

Polymerase chain reaction enzyme

Deep sea hydrothermal vent bacterium

Recombinant protein

Fomulaid (Market Bio-seiencess, Columbia, MD)

Fatty acids used as additive in infant formula nutritional supplement

Marine microalgae Cell culture

Acquorin Bioluminescent calcium indicator

Bio luminescent jellyfish, acquora victora

Recombinant protein

Green Fluoresecent Protein (GFP)

Reporter gene Bio luminescent jellyfish, acquora victora

Recombinant protein

Phycoerythrin Conjugated antibodies used in ELISAs and flow cytometry

Red algae Cell culture

Resilience Marine extract additive in skin creams

Caribbean gorgonian, Pseudopterogorgta elisabethea

Wild harvest of gorgonian

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Trabectedin (Yondelis, ecteinasicidin-743, ET-743): The marine alkaloid ecteinascidin

743 (ET-743) is by far the most advanced anticancer compound which belongs to the

tetrahydroisoquinoline alkaloid class. It is produced from the ascidian Ecteinascidia

turbinata. This is approved by the EMEA in September 2007 for the treatment74,75 of

advanced soft tissue sarcoma. ET-743 is in late stages of phase III clinical trials for the

treatment of ovarian cancer (with Johnson & Johnson in the US) and other ongoing Phase

II trials include paediatric sarcomas, breast and prostate cancers. The alkaloid was shown

to be a minor-groove alkylator of DNA and disrupts cell cycle causing cell proliferation

inhibition and to cause inhibition of MDR1 gene transcription47 the latter being

responsible for the well-known phenomenon of multi-drug resistance (MDR), which

causes tumors to become insensitive to anticancer drugs and is a severe obstacle for

chemotherapy. ET-743 furthermore elicits non-p53-mediated apoptosis in tumor cells.

One of the predominant toxicities exhibited by ET-743 in preclinical studies was

hepatotoxicity, particularly in the female rat, and similar effects had been seen in human

patients but could be controlled by dose-reduction. However, in recent publication,

Donald et al.76 demonstrated that pretreatment with high-dose dexamethasone gave

complete protection against hepatotoxicity in this animal. Thus such a treatment in

humans may well be a method of controlling this Et-743 –related toxic side effect. This is

produced commercially by semi-synthesis from the eubacterium derived

cyanosafracin B.77

Didemnin B: Didemnins provoked interest way back in the 1980s due to their

pronounced anti-tumour activity because of their interference with the protein synthesis.58

14

Its further development as anticancer drug was recently abandoned, after Phase II trials,

due to its hepatotoxic side effects.

Aplidine: Dihydrodidemnin B (Aplidine) appears to be less toxic and even 5 to 6 times

more effective than didemnin B with a broad spectrum activity both in vitro and in vivo

against various types of cancer diseases such as colorectal, lymphoma, thyroid and renal

cancers. 59 It further shows anti-angiogenic activity in experimental models. Phase II

clinical trials are underway in Europe for renal and colon carcinomas. European

Commission has approved aplidine for acute lymphoblastic leukaemia and other trials

covering renal, head and neck, and medullary thyroid are ongoing, simultaneously.60

Phase II clinical trials are ongoing by Pharma Mar.78-80

Kahalalide F belongs to the family of dehydroaminobutyric acid containing cyclic

peptides isolated from the Hawaiin mollusk, Elysia rufences. It has shown antitumor

activity probably by interfering with lysosome function in prostate, colorectal and lung

cancer cell lines.60,61 It is currently in phase II trials for prostate cancer and other solid

tumours.81-84

Bryostatins are macrocyclic lactones isolated from the marine bryozoan Bugula naritina.

Bryostatin 1 is one of the most abundant and best-studied compounds of this series and is

found to have anticancer and immunostimulating activities. Bryostatin 1 binds to protein

kinase C with high affinity, which may be the mechanistic basis for both observed

anticancer and immunostimulatory activities. Since the publication of the first structure

by Pettit in 1982, these molecules have been the target of many synthetic groups but only

15

three of the 20 reported bryostatins have been synthesized. It is presently in Phase I/II

clinical trials.85-89

Halichondrin B: (eribulin (E-7389, NSC-707389) is one of the series of compounds

originally isolated and reported by Umera et al. in 1985 from the Japanese sponge

Halichondria okadai. It is a tubulin interactive agent, affecting tubulin depolymerization

at a site close to, but distinct from the Vinca site. It is currently in Phase II/III clinical

trials.90-96

Dolastatin-15: (Synthadotin, ILX-651) This is an orally active third generation dolastatin

derivative that was licensed by Iiex from BASF Pharma. It is currently in phase II clinical

trials by Genzyme.97-99

The other areas in which considerable development has been made are pain and

inflammation. It is in this area where conotoxins have gained their importance.

Conotoxins are a group of peptides produced by shells, which have a variety of biological

actions.

-Conotoxin MVIIA: (SNX-III) This 25-residue peptide with three interlocking

cystinyl bridges was originally isolated by Olivera. The pain-killing marine natural

product has successfully completed Phase III clinical trials for two applications: to

alleviate pain associated with malignant diseases (Cancer and AIDS) and as an analgesic

for nonmalignant neuropathic pain.42

Ziconotide: (Prialt) This is a semisynthetic analogue of conotoxin with a 25 amino acid

peptide exhibiting three disulfide bonds. It is an N-type calcium channel -conotoxin

MVIIA. This compound is launched by Elan in both US and Europe in 2005 for the

16

treatment of patients suffering from chronic pain.100 In March 2006 Eisai obtained the

rights to market ziconitide in Europe. The compound proved to be 1,000 times more

active than morphine in animal models of nociceptic pain. The activity of ziconotide is

due to the blockage of N-type calcium channels and thereby inhibits the release of

neurotransmitter. In contrast to morphine, it does not induce the development of

tolerance, constipation or respiratory depression. Following the discovery of this Conus

peptide in the late 1970s, no efforts were spared to develop more potent synthetic

analogues. US FDA approved ziconotide for hard-to-treat pain associated with cancer,

AIDS and neuropathies.

Xen-2174: This conotoxin is undergoing clinical evaluation, as all the other conotoxins

have been halted or discontinued. Xen-2174 a 13-aminoacid peptide with two cysteine

bridges isolated from Conus marmoreus has been found to inhibit the norepinephrine

transporter (NET), a known CNS drug target that is inhibited by the antidepressant

desipramine.101-103 Currently Xen-2174 is in Phase I/II trials for the treatment of cancer

pain.104 Other conotoxins which have been halted or discontinued are contulakin G

(CGX-1160) and conotoxin – G (CGX-1007).

Methopterosin (OAS 1000): The extracts of Caribbean gorgonian Pseudopterogorgia

elizabethae showed anti-inflammatory activity and are nowadays used as an ingredient

for cosmetic skin care products.105 The activity is due to the diterpene glycosides-

pseudopterosins-which inhibit phospholipase A2. Methopterosin (OAS-1000), a

derivative of pseudopterosin, is currently under Phase I trials.

17

IPL 576: A synthetic analog of the steroid contignasterol isolated from the sponge

Petrosia contignata,106-108 it is in phase II trials as a leukocyte-suppressing anti-

inflammatory drug for the treatment of asthma.

GTS 21: 3-(2,4-dimethoxy benzylidene)-Anabaseine, is a selective 7-nicotinic

acetylcholine receptor partial agonist in clinical development at Taiho to treat

Alzheimer’s disease and schizophrenia.109

Trodusquemine (MSI-1436): This is a sulfated aminosterol from the dog fish shark

Squalus acanthias along with the closely related squalamine and other steroids.110

Squalamine has been shown to inhibit endothelial cell proliferation but clinical trials in

cystic fibrosis and oncology were halted in July 2007. In contrast to squalamine,

trodusquemine suppresses mammalian appetite through inhibition of protein tyrosine

phosphatase 1B.111-113

18

N

N

N

N

NH2

Ara - A

OHO

HOOH

N

S

O

HOOCCH(CH2)3CN H

COOHCH2OCO CH 3

NH2

O

Cephalosporin C

HN

N

OR

O

Spongouri d ine R = MeSpongot hym idine R = H

Ma it o t o x in

O O

O O

O

O

OH

O

O

O

OOH

OH

O O

O O

OO

OOH

HO

O

OOH

O

OO

O

O

O

OO

O

O

O

OO

OHOSO3Na

OH

OHHO OH

HO

OH OH

OHNaO3SOHO OH

OH

OHOH

HOOH OH OH

OH

OH

HO

OH

O O

NH

OH

O

O

O NH

O

NNMe

OO

N

ONMe

O

NR

O

OMe

H

Didem nin B R = CH(OH)CH3Dehydrodidem n in B (Apl id ine)

R = COCH3

NNMe

O S

NH

OOH

OMeHIO

OAc

OO

MeO

HO

H

Ec t einasc idin 743

K RN 7000

O

OH

OH

OHO

OH

(CH2)13CH3

NH

(CH2)24CH3O

OH

OH

HOO

HOOH

ThrGlySerCysArg

SerGly

Cys Cys Asp TyrMet

LeuArgSerCys

Lys

Ala Gly

LysGlyLysCysH2N

Lys

CysH2N

Ziconotide

19

5-MeHex

Val-5

NH

NH

N

O

NHO N

O

H2N

NH

O

O

HNO

NHN

OO

N

O

NHO

O

HONH

NH

O

O

H

H

H

L-OrnD-allolle-2

Thr-1

D-allolle-1

Val-2

L-PheThr-2

Val-3

L-Pro

Z-Dhb

Val-1

Val-4

Kahalalide F

Methopterosin

Me

OOH

Me

HMe

O

OHHO

OH

RSqualamine H

Trodusquemine

NNNH OH

OSO3-

R

H2N NH

NN

OMe

OMe

E

.2 HCl

GTS-21

O

O

OO O

O

O

OOO

HH

HH

HH

OH

H

H H H

H

OH

H

H HH

H

O

O HO

OHOOH OH

HH

Halichondrin B

Discodermolide

OO H

OH

OH OCONH2

HO

OH

20

A major issue that has to be considered when a marine chemical entity is selected

for clinical development is sustainable and industrially feasible supply of the material.

A crucial step is the incorporation of a sustainable supply, which has delayed the

development of these agents. Marine organisms for drug discovery research have been

collected using various methods such as scuba diving, submersibles, dredging and

trawling. Submersibles (Fig. I-02) enable scientists, to access unusual habitats, such as

vent communities and deep-sea benthic habitats.

Fig. 1-02: Submersible

However, the metabolites occur in trace amounts in the organism and a steady source of

supply from wild harvest can’t provide enough of the target compound for the

development studies.

The concentrations of many highly active compounds in marine invertebrates are

often minute, sometimes accounting for less than 10-6 % of the wet weight. ET-743 is the

best example to illustrate this problem. For example, in order to obtain approximately 1 g

of the promising anti-cancer agent ET-743, close to 1 metric tonne of the tunicate

21

Ecteinascidia turbinata has to be harvested and extracted. Scientists at PharmaMar

performed an elegant semisyntheses from the marine Pseudomonas fluorescens

metabolite cyanosafracin B that provided cGMP grade ET-743 from a 21-step synthetic

process on a scale large enough to provide enough material for clinical trials. This was

feasible despite a low overall yield of 1.4% because the starting material could be

obtained on a large scale by fermentation.46

Various approaches to overcome this problem are

1. Chemical synthesis

2. Controlled harvesting

3. Mari-culture: Favoring the growth of the organism in its natural milieu.

4. Aquaculture: Culture of the organism under artificial conditions.

5. Genetic intervention (Cloning)

6. Semi synthesis: Use of a parent/related compound as the starting point

followed by a short / industrially effective synthetic process.

Marine invertebrates are laden with bacterial symbionts, often in high density.

Because many marine natural products structurally resemble bacterial compounds, it has

long been proposed that the marine chemicals are produced by bacterial symbionts.

Based on the symbiotic hypothesis of marine natural products synthesis, two basic

strategies can be used to supply for development. First, bacteria can be cultured from

these organisms followed by detection of the compound in the culture. Second, genes for

the biosynthesis of important molecules can be cloned and heterologously expressed.

Recently Hildebrand et al. 116 reported the cloning and partial sequencing of the putative

bryostatin biosynthetic gene Bry A which contains the required functionality to make

22

most of the important ‘recognition domain’ portion of the bryostatin, potentially enabling

production of this portion via genetic engineering. In combination with chemical

synthesis or further genetics this could provide the first example of a symbiotic natural

product supplied by the genetic approach.

Both the sea and land-based aquaculture methods can be considered as an

alternative to harvesting wild specimens. Bugula neritina, the source of bryostatins and

Ecteinasicidia turbinata, the source of ecteinascidin-743 have been produced under

controlled conditions by Cal Bio Marine Technologies 117 (Carlsbad, CA) (Fig. I-03 and

Fig. I-04)

Fig. I-03: Aquacultured growth plate of Bugula neritina

Fig. I-04: Aquacultured colony of Ecteinascidia turbinata

23

Table I-02: Potential Therapeutic Compounds isolated from Marine Organisms

Source Compound Disease area Status References Tunicata

Ecteinascidia turbinata Ecteinascidin 743 Cancer Phase III

74,75,77

Aplidium albicans Dehydrodidemnin B (Aplidine)

Cancer Phase II 78-80

Bryozoa Bugula neritina Bryostatin 1 Cancer Phase II 85-89 Elysia rubefscens Kahalalide F Cancer Phase I 80-83 Molluscans Conus magnus Ziconotide Chronic pain Phase III 93

Dolabella auricularia Dolastatin 10 Cancer

Development discontinued

by NIH

114

Dolabella auricularia Dolastatin 15 Cancer

Phase II under preparation

97-99

Porifera (sponges) Halichondria okadai Halichondrin B Cancer Phase II / III 90-96

Hemiasterella minor Hemiasterlin (E 7974)

Cancer Phase I 115

Discodermia dissoluta Discodermolide Cancer

Discontinued due to toxicity

114

Agelas mauritiamus KRN 7000 Cancer

Discontinued due to

cytotoxin antagonism

114

Pseudopterogorgia elizabethae Methopterosin

Wound healing/

Inflammation Phase I

105

Petrosia contignata IPL576902 (11) Inflammation/

Asthma Phase II

106-108

Nemertia Amphiponus lactifloreus GTS-21

Alzheimer’s/ schizophrenia

Phase I 109

Others

Spisula polynmya Spisulosine Cancer Discontinued

due to lack of efficacy

114

24

Squalus acanthias Trodusquemine Antihyperlipi

demic, Antidiabetic

110-113

Squalus acanthias Squalamine Cancer Trials

discontinued 114

Conceptually, it is clear that the marine ecosystem offers a huge potential in the

naturally based pharmacopoeia of this century. However, an unfavorable balance between

discovery and the very small number of candidates incorporated for clinical evaluation

exists. So it appears that a better and more pragmatic approach is urgently needed in

order to translate innovative discoveries into active clinical therapeutics.

25

Present research work of the author

India is surrounded on three sides by oceanic waters especially in near tropical or

tropical zones thereby harbouring innumerable genera and species of marine plants and

animals. A few research groups in India have been engaged in these efforts on marine

metabolites with assistance from government funding agencies. One of the prominent

groups under the leadership of Prof. Ch. Bheemasankara Rao and Prof. D. Venkata Rao

has been pursuing research on bioactive metabolites from sea organisms of the Indian

Ocean for the past two decades. The author who is part of this investigative group had

gone through extensive literature in this area and is much fascinated to put his efforts in

the investigation. After a preliminary screening the following organisms were chosen for

extensive chemical investigations

Dendrilla nigra Dendy (Sponge)

Hyatella cribriformis Hyatt (Sponge)

Synoicum indicum (Ascidian)

The author isolated 3,4 - diaryl pyrrole alkaloids, phenolic compounds,

sesterterpene with interesting scalarane skeleton, sphingolipids, polyaromatic brominated

phenolic compounds. During the study of 3,4-diaryl pyrrole alkaloids, the author has

come across extremely interesting data on this group of compounds from marine

organisms and preferred to incorporate a detailed account on this class of compounds in

the following pages.

26

3, 4-DIARYL PYRROLE ALKALOIDS

Marine ascidians or tunicates and sponges are prominent producers of metabolites

derived from amino acids. The amino acid DOPA (2-amino, 3- (3’, 4’– dihydroxy

phenyl) propionic acid) in particular, appears to play an important role in the metabolism

of these marine invertebrates, serving as the apparent precursor of several alkaloidal

metabolites isolated from this source. DOPA metabolic products range from peptides to

polycyclic alkaloids. Examples of peptide products are the tetrapeptides, halocyamines

and the tripeptides known as tunichromes.

Alkaloids apparently derived via DOPA metabolism include the lamellarins,

ningalins, polycitrins, lukianols, purpurone, storniamides, dictyodendrins, all of them

having a central pyrrole ring usually with 3,4- diaryl substitutions.

O

N

R1R1R1

R1

R1

O

Lamellarins R1=H or Me R2=H,OMe or OH R3=H or OH

R2

R1

R3

N COOMe

HO OH

OMe

RO

Lamellarins O and P R=H or OH

N

BrHO

Br

Br OR

Br

Polycitrins R=H or Me

OO

OH

N

HO

R

OH

Lukianols R=H or I

O

OH

O

NHN

HOR2

HN

OHR3

OHHOHO

OH OHR1

OH

O O

Storniamides A R1=OH R2=R3=H B R1=R3=OH R2=H C R1=H R2=R3=OH D R1= R2=R3=OH

NH

OO

O O

HO OH HOOH

Ningalin A

27

N

HO OH HO OH

Purpurone

O O

HO

HO

HO

OH

OH

N

HO

Dictyodendrin AOH

NH

OSO3Na

OH

OH

MeOOC

DOPA – derived metabolites are isolated from many ascidians which accumulate

massive quantities of ionic vanadium and iron from seawater and suggested to participate

in this process. In didemnid ascidians vanadium accumulation is negligible, but iron

accumulation is a prominent feature generating in vivo iron concentrations up to 107

times higher than that found in seawater. While an understanding of this process appears

incomplete, the metal binding phenomenon has been partially explained by complexation

with O-catechol functionalities. The mechanism involves chelation of metals such as

Fe3+ and V5+ by dopa residues.

Lamellarins:

Lamellarins are first isolated from the prosobranch mollusk Lamellaria sp. in

1985.118 They were later obtained from the ascidians Didemnum chartaceum 137

(Seychelles ascidian) Didemnum sp. 150,168 (a Great Barrier Reef ascidian) and the sponge

Dendrilla cactos.161,162 Sulfated lamellarins 169 were also isolated from an unidentified

ascidian collected from the Arabian Sea.

Lamellarins fall into three structural groups depending on whether the central

pyrrole ring is fused (lamellarins A-N) or unfused (lamellarins O-R) to adjacent aromatic

rings and on the presence between atoms 5 and 6 of the quinoline moiety of either a

single(lamellarins I-L) or a double bond (lamellarins B, D, M,N).

28

The first four lamellarin alkaloids, 118 lamellarins A (1), B (2), C (3), and D (4)

were reported from the mollusk Lamellaria species collected near Koror, Palau. The

structure of lamellarin A was determined by an X-ray crystallographic study and the

structures B-D were assigned by interpretation of spectral data. Lamellarin A exists as a

1:1 mixture of two geometrical isomers due to restricted rotation about C1-C11 bond. At

concentration of 19μg/ml, lamellarin D caused 78% inhibition of cell division in the

fertilized sea urchin egg assay while lamellarin C caused 15% inhibition and lamellarins

A and B were inactive.

The first total synthesis of lamellarins D and H was accomplished by using N-

ylide mediated pyrrole ring formation and subsequent lactonization. 119 In 2002, Ishibashi

et al. 120 synthesized ten derivatives of lamellarin D and evaluated them for cytotoxicity

against a Hela cell line in an effort to examine their structure activity relationship. It

appeared that the hydroxyl groups at positions C-8 and C-20 of lamellarin-D were

important structural requirements for cytotoxic activity, while the hydroxyl group at C-14

and the two methoxy groups at C-13 and C-21were not necessary for the activity.

Tardy et al. 121 exploited the lamellarin D pharmacophore for the development of

topoisomerase I-targeted anticancer agents. Lamellarin D is a potent poison of human

topoisomerase I endowed with remarkable cytotoxic activities against tumor cells and

potentially stabilizes topoisomerase1-DNA covalent complexes so as to promote the

formation of DNA single strand breaks. The 5-6 double bond is an essential element for

the poisoning of topoisomerase I and the antiproliferative activity. The three phenolic OH

groups at positions 8, 14 and 20 are important structural elements but they can be

29

substituted without the loss of activity and the cationic proline and valine derivatives at

these positions are selected for preclinical development.

In 2005, an efficient and highly convergent total synthetic route to lamellarin D

was achieved in 8 steps. 122

Vanhuyse et al. 123 reported that lamellarin D is a potent apoptic agent and its

cytotoxic action is fully maintained in multidrug resistant cells compared to the sensitive

parental cell line. The multidrug resistance transporter proteins P-glycoprotein, multidrug

resistance protein and lung resistance protein have been associated with treatment failure.

They are plasma membrane transporters responsible, at least in part, for the resistance of

tumor cells to chemically and/or functionally unrelated drugs. It is therefore important to

identify new anticancer agents that are not sensitive to these export pumps. Lamellarin D

is found to be equally toxic to murine leukemia P388 cells and its camptothecin resistant

cells P388CPT5 expressing a functional P glycoprotein. These results are interesting and

tend to reinforce the potential of this marine alkaloid as a lead compound to design novel

topoisomerase 1 targeted anticancer agents.

Iwao et al. reported the synthesis of Lamellarin D, L and N.124 A library of open

lactone analogues of lamellarin D was prepared from the scaffold of methyl 5,6-dihydro-

pyrrolo [2,1-a] isoquinoline-3-carboxylate by introducing various aryl groups through

sequential and regioselective bromination followed by Pd(o)-catalyzed Suzuki cross

coupling chemistry. The SAR study concluded that more than 75% of the open chain

lamellarin D analogues tested showed cytotoxicity in a low micromolar GI50 range. 125

30

Iwao et al. 126 reported an efficient route for the synthesis of 1-dearyllamellarin D

using directed lithiation, Suzuki-Miyaura coupling and palladium–catalyzed direct

arylation as the key reactions.

Albericio and Alvarez reported the design and synthesis of poly (ethylene glycol)

derivatives of lamellarin D with the aim of modulating their physicochemical properties

and improving the biological activity. Mono-, di-, and tri-PEG conjugates with improved

solubility were obtained from corresponding partially protected phenolic derivatives of

lamellarin D. Conjugates were evaluated for their cytotoxicity. 127

Alvarez et al. 128 reported the design and synthesis of Lamellarin D conjugates

with a nuclear localization signal peptide and a poly (ethylene glycol)-based dendrimer.

Conjugates were obtained from the corresponding protected lamellarin D. Conjugates

were found to be more cytotoxic than the parent compound.

Hu et al. 129 reported their work on developing 1,2-diphenyl-5,6-dihydropyrrolo

[2,1-a] isoquinolines as hybrids of combretastatin A4 and lamellarin D with the aim of

retaining the cytotoxicity and antimitotic activities of the parent compound. Ruchirawat

et al. prepared Lamellarin D analogues containing a lactam rather than a lactone. Their

work was the first report of a SAR studies done for a change at this position. 130

In late 2010, Li et al. 131 reported a new, concise synthesis of lamellarin D and

lamellarin H in seven steps.

Recent advances in the pharmacological development of Lamellarin D include

the identification of new cellular targets of this compound and new insights in to its

mechanism of action. The maleate-aspartate shuttle was recently identified as a new

31

target for Lamellarin D by proton NMR- based metabolomic. 132 Certain protein kinases

relevant to cancer have recently been reported as targets of Lamellarin D, Lamellarin N

and other lamellarins. 133 Lamellarin D exerts its anti-tumor activity through

complementary pathways: a nuclear route via Topoisomerase I (Topo I) inhibition 134,135

and mitochondrial targeting by induction of mitochondrial permeability transition (MPT).

136

In 1988 Lindquist and Fenical 137 reported four new lamellarin class alkaloids,

lamellarins E (5), F (6), G (7) and H (8) from the marine ascidian Didemnum chartaceum

of the Indian Ocean. The structure of lamellarin E was determined by spectroscopic and

X-ray crystallographic methods. The structures of lamellarins F-H were elucidated by

interpretation of NMR spectral data, which relied heavily upon JC-H and 2-3JC-H correlation

experiments.

Prosobranch mollusks of the family Lamellariidae have been described as specific

predators of colonial ascidians. 138-140 These mollusks preying on ascidians appear to have

followed a similar evolutionary pattern as the numerous opisthobranchs preying on

chemically rich sponges, bryozoans, coelenterates and algae. The report of lamellarins E-

H from D.chartaceum indicates that Lamellaria species most likely acquired lamellarins

A-D from a similar ascidian food source.

In 1997, Steglich’s group described a biomimetic synthesis of lamellarin G

trimethyl ether. 141 In 2001, Ruchirawat et al. reported the efficient synthesis of lamellarin

G trimethyl ether. The synthesis involved formation of the core pyrrole-[2, 1-α]

isoquinoline followed by formation of the lactone ring. 142 In 2003, Iwao et al. reported

32

the synthesis of lamellarin G trimethyl ether starting from a symmetric 3,4-dihydroxy

pyrrole bis-triflate derivative. 143

Handy et al. reported a modular synthesis of the lamellarin G trimethyl ether that

was based on application of three iterative sequential and regioselective halogenation /

Suzuki cross coupling events. 144 In 2006, lamellarin G was synthesized by the key step

of formation of 3,4-diarylpyrrole-2,5-dicarboxylic acids from arylpyruvic acids and 2-

arylethyl amines. 145

Opatz and Liermann reported a convergent synthesis of lamellarin U and

lamellarin G trimethyl ether from a readily available deprotonated α-aminonitrile as an

AB ring building block. 146 Gupton et al. reported formal synthesis of Lamellarin G

trimethyl ether and ningalin B via formation of the polysubstituted pyrrole derivatives,

from a vinylogous iminium salt derivative. 147

In 2005, lamellarin H and its derivatives were synthesized by using the fabricated

intermediate, 2,4,5-trimethoxy-α-chloroacetophenone as key starting material for this

synthesis. 148 In 2006, the same group reported the synthesis of the second intermediate of

lamellarin H, 1-(3,4-dimethoxy phenyl)-8,9-dimethoxy-2-(2,4,5-trimethoxyphenyl)-

pyrrole-[2,1-α]-isoquinoline. 149

An Australian Didemnum species has yielded six new alkaloids 150 lamellarins I

(9), J (10), K (11), L (12), M (13) and N (14) together with lamellarins A to D that were

previously obtained from the prosobranch mollusk Lamellaria species. These structures

were deduced using chemical and spectroscopic methods. Lamellarin K and L exhibit

immunomodulatory effects. 151

33

Lamellarin K has been synthesised using an intramolecular (3+2) cycloaddition

reaction to form the central pyrrole ring. 152 In 2001 lamellarin I and K were synthesized

based on a [3+2] cycloaddition. 153 In 2006, Steglich et al. developed biomimetic

synthesis of lamellarin K in four steps. 154

Lamellarin L has been synthesized by using biometric strategy. 155 In 2000,

Steglich et al. developed biomimetic total synthesis of the non symmetrical lamellarin L

in five steps. 156 Lamellarin L and U were generated via a total solid phase synthesis.

157,158 In 2004, Ruchirawat et al. obtained lamellarins K and L in three steps. 159 In 2006,

Iwao et al. synthesized lamellarin L. 160

Lamellarins O (15) and P (16) were isolated from the sponge Dendrilla cactos

that was obtained by dredging off South Australia. 161 The next two lamellarins Q (17)

and R (18) were isolated from a recollection of the Dendrilla cactos from New South

Wales. 162

Lamellarins O and Q were synthesized using Stille, Suzuki, and Negishi cross-

coupling reactions as the key steps. 163 Boger et al reported the total synthesis of

lamellarin O, utilizing a common heterocyclic azadiene Diels- Alder reaction. 164 In 2004,

Albericio’s group described an efficient solid-phase strategy for synthesis of lamellarins

O and Q using Merrifield resin and N-protected methyl 3, 4-dibromopyrrole-2-

carboxylate as a scaffold. 165

The total syntheses of lamellarins O, P, Q and R have been achieved by using

cross-coupling and the directed lithiation as key reactions. 166

34

In 2007, Liu et al. reported 167 four new lamellarin-like phenolic pyrroles,

neolamellarin A (19), neolamellarin B (20), 5-hydroxy neolamellarin B (21) and 7-

hydroxy neolamellarin A (22) from the marine sponge Dendrilla nigra which was

collected from shallow water in Saipan. These structures closely resemble the structure of

the known Dendrilla cactos compound lamellarin O (15). Compound (22) inhibited

hypoxia induced HIF-1 activation in T47D cells. Hypoxia induction of vascular

endothelial growth factor (VEGF), a potent angiogenic factor and HIF-1 target gene, was

also inhibited by (22) at the secreted protein level.

Lamellarin S (23) was isolated from a south – eastern Australia tunicate of the

genus Didemnum. 168

An unidentified ascidian from the Arabian Sea (Trivandrum Coast of India)

yielded nine new alkaloids of the lamellarin class T (24), U (25), V (26), W (27), X (28)

and Y (29) together with lamellarin N. Lamellarins T, U, V and Y were obtained as 20-

sulfated derivatives. 169 This is the first report of lamellarin sulfates.

In 2006, Banwell et al. reported the synthesis of lamellarin T hybrids and were

evaluated for anti-mitotic and cytotoxic properties. The key steps include selective

lithium - for halogen exchange, Negishi, and Suzuki-Miyaura cross-coupling reaction. 170

Davis et al. 171 reported 20-sulfated derivatives of lamellarins B, C and L, the 8-

sulfated derivative of lamellarin G and also the lamellarin Z (30). Lamellarin G 8- sulfate

is the first example of this class of compounds sulfated at C-8 position, while lamellarin

Z is the first example of dimethoxylated lamellarin. An aberration in the integration of

signals in the 1H NMR spectra of the 20-sulfated derivatives of lamellarins B, C and L

35

led to NMR relaxation studies.T1 ( relaxation time) values were calculated for all protons

in the sulfated lamellarins and their corresponding non sulfated derivatives. Interestingly

the protons ortho to the sulfate group have T1 values up to five times larger than the

corresponding protons in their non sulfated derivatives. These are due to the isolation of

these protons which have minimal relaxation pathways.

Rami Reddy et al. 172 reported lamellarin α 20-sulfate (31) from an unidentified

ascidian collected from the Arabian Sea near Trivandrum, India. They also screened

lamellarins for HIV-1 integrase inhibitory activity and found them showing selective

inhibition. Of all the lamellarins, lamellarin α 20-sulfate significantly inhibits integrase

protein in vitro and HIV viral replication in cultured cells. Lamellarin α 20-sulfate acts in

a part of the viral life cycle consistent with inhibition of integration.

These findings provide a new class of compounds for potential development of

clinically useful HIV integrase inhibitors. Embedded within the lamellarins is a coumarin

moiety. Coumarins have been found to be active as integrase inhibitors. Sulfated

compounds have also been found to be inhibitory in some cases.

In 2006, the first total synthesis of lamellarin α 20-sulphate was developed in 14

steps. 173

Ham and Kang 174 reported lamellarin β (32) from a purple unidentified

Didemnum species collected in the Indian Ocean. This alkaloid showed cytotoxicity

against human promyelocytic leukemia HL-60 with an IC50 of 4.8 μg/ml.

Lamellarins γ (33), and ε (34) along with eight known lamellarin alkaloids M,

K, K-diacetate, K – triacetate, U, I, C–diacetate and X – triacetate were isolated from the

36

Indian ascidian Didemnum obscurum. 175 The structures were established using standard

spectroscopic techniques. Lamellarins are cytotoxic and showed good antitumor activity.

Many antioxidant compounds possess anticancer properties. Hence the lamellarin

compounds were screened for anti oxidant activity. The absence of 8-OH resulted in the

decreased potency of anti oxidant activity and 14 -OH is not necessary for the activity.

Reddy et al. 176 reported lamellarin - (35), lamellarin-(36), lamellarin - (37),

lamellarin - (38) along with seven known lamellarins, lamellarin K, I, J, K–triacetate

lamellarin L-triacetate, F, T-diacetate and they also screened for cytotoxic activity against

colorectal cancer cells (COLO - 205). Lamellarin - and , L–triacetate and F have

shown excellent activity against test cancer cell lines.

To establish a more comprehensive structure activity relationship relatively large

quantities of lamellarins are required. However, because the natural sources of

lamellarins, some species of ascidians, sponges, and mollusks, provide these compounds

in only minute quantities, total synthesis is a vital alternative in providing these

compounds for detailed biological evaluations. Ploypradith et al. 177 reported four

polymer-supported reagents which are utilized in multi-step synthesis of lamellarins.

1. Amberlyst A-26Br3- 2. Polymer bound pyridine hydrobromide perbromide

(PVPHP), 3.Amberlyst A-26 NaCO3- 4. Amberlyst – 15

Marco et al. 178 compared the lamellarin activities with camptothecin. Eukaryotic

topoisomerase is the target for the anticancer drug camptothecin. They have built

molecular models of the ternary complexes formed between the DNA Top1 ensemble and

lamellarin D or camptothecin fully intercalated in to the duplex DNA and studied by

37

means of nanosecond molecular dynamics simulations in aqueous solutions. Results

showed that 20-OH and 8-OH of lamellarin D participated in hydrogen-bonding

interactions with the side chains of Glu 356 and Asn 722. It was also found that

lamellarin D stabilizes Top1 cleavage at CG sites in addition to TG sites observed for

camptothecin. They also confirmed the deleterious effect of replacing the 20-OH in

lamellarin D with hydrogen using a set of thermodynamic integration free energy

simulations.

Ruchirawat et al. synthesized 28 natural (lamellarins C,E,F,G,I,J,K,L,T,U,Yand χ)

and unnatural lamellarins with either a saturated or an unsaturated D ring. 179

Thipnate et al. studied lamellarins using receptor independent (R1) 4D

quantitative SAR (QSAR models). 180,181 They obtained valuable 3D pharmacophore

information from a set of 25 structurally complex lamellarins screened insilico against

human hormone dependent T47D breast cancer cells. Overall, they identified formation

of an intermolecular hydrogen bond and the hydrophobic interactions of substituents at

C-10, C-11 and C-12, as the most important features for cytotoxicity against the cancer

cells. They also suggested that hydrophobic substitutions at C-3’ and C-4’ could enhance

cytotoxicity.

Finally, lamellarins not only have interesting structural features, but also exhibit a

wide array of significant biological activities, including cell division inhibition,

cytotoxicity, HIV-1integrase inhibition, antioxidant activity and immunomodulatory

activity and they definitely provide a new class of compounds for potential development

of clinically useful agents.

38

Further studies can contribute to a better understanding of the mechanism of

lamellarins and their analogues and will be beneficial for the ongoing pharmacological

optimization of this class of compounds.

Pyrrole-Derived Alkaloids Related to Lamellarins:

Ningalins:

Four DOPA – derived alkaloids Ningalins A (39), B (40), C (41) and D (42) three

of which possess new carbon skeletons were isolated from a western Australian ascidian

of the genus Didemnum. 182 The structures were elucidated by interpretation of overall

spectral data and by 2DNMR correlation methods. Ningalins A-D are composed of C18,

C25, C32 and C40 condensed aromatic systems with the unifying theme that all are derived

via the condensation DOPA.

Ningalin A is synthesized using Diels-Alder strategy. 183 Ningalin B is synthesized

and shown to be a multi-drug resistant reversal agent. 184 In 2002, Bullington et al.

reported the synthesis of ningalin B. 185 A formal synthesis of ningalin B was reported in

2003. 186 Gupton et al. reported the application of vinylogous iminium salt derivatives to

a convenient and efficient synthesis of ningalin B hexamethylether. 187 Steglich et al.

reported a biomimetic synthesis of ningalin B. 188

Peschko et al. reported the synthesis of the ningalin C. 189 In 2005, Boger et al.

reported the synthesis of ningalin D in nine steps. 190

Ningalins possess remarkable multidrug resistance (MDR) modifier activity. Key

analogue derivatives of ningalins were examined resulting in the discovery of a potent

39

MDR reversal agent that hypersensitizes P-glycoprotein resistant tumor cell lines to front

line conventional therapeutic agents. 191

Ningalin derivatives have been evaluated for their properties of potent reversal of

MDR and use in drug combinations against human colon carcinoma xenograft in nude

mice. 192

Storniamides:

Storniamides A (43), B (44), C (45), and D (46) were isolated from a sponge

Cliona species collected near San Antonio Oeste, Rio Negro, and Argentina. 193 They

showed antibiotic activity against Gram-positive bacteria (Staphylococcus aureus,

Bacillus subtilis and Micrococcus luteus) at 50 μg/disk. Five tyrosine units take part in

the building of the structural frame work of the storniamide, while subsequent

hydroxylation in the aromatic rings gives rise to different compounds.

Boger et al. reported the total synthesis of permethyl storniamide A. 183 In 2003 a

formal synthesis of permethyl storniamide A was reported, which was based on a highly

efficient route to 3,4-diarylpyrrole marine alkaloids. 186

In 2002 Furstuner et al. 194 made an assessment of the DNA cleaving properties of

the pyrrole alkaloid derivatives permethyl storniamides, lamellarins, ningalins etc. They

concluded that an increase in the number of peripheral methylations of such pyrrole

alkaloids causes a sharp decrease in their antitumoral activity.

40

Polycitrins:

Polycitone A (47) and polycitrins A (49) and B (50) were isolated from the

marine ascidian Polycitor species. 195 The penta-O-methyl derivative of polycitone A

was found to inhibit the growth of SV40 transformed fibroblast cells in a concentration of

10 ug/ml. Polycitone A inhibited the RDDP and DDDP activities of HIV-1 RT. 196

Polycitone B (48) was isolated from the ascidian Polycitor africanus from

Madagascar. 197

In 1995, Steglich et al. described the biomimetic total synthesis of polycitrin A. 198

The same group reported shorter synthetic route to polycitrin A in three steps. 199 Beccali

et al. reported the synthesis of Polycitrin B. 200 In 2002, Steglich’s group first synthesized

both Polycitones A and B. 201 In 2006, Gupton’s group demonstrated a new and efficient

relay strategy to synthesize Polycitones A and B. 202

Purpurone:

Purpurone (51) was isolated from the marine sponge Iotrochota species. 203 Its

structure was established mainly on the basis of NMR spectroscopic data. Purpurone is

having ATP-citrate lyase inhibitory activity. ATP-citrate lyase inhibition is anticipated to

reduce the production of acetyl CoA and can affect both lipogenesis and

cholesterogenesis, and can be a strategic target for hypercholesterolemia therapy.

Purpurone is synthesized by Peschko and Steglich 204 in 2000. Jia et al. 205

reported the direct synthesis of polysubstituted pyrroles from readily available aldehydes

and amines and their application to the total synthesis of purpurone.

41

Fifteen new purpurone related alkaloids named baculiferins A-O (52-66), were

isolated from the Chinese marine sponge Iotrochota baculifera, 206 together with the

known alkaloids purpurone (51) and ningalin A(39). Most of the new compounds contain

one to three O-sulfate units. Their structures were determined by extensive spectroscopic

analysis including 1H and 13C NMR (COSY, HMQC, HMBC) and ESIMS data.

Baculiferins were found to possess anti-HIV-1 activity.

Lukianols:

Lukianols A (67) and B (68) were isolated from an unidentified encrusting

tunicate 207 from Palmyra Atoll and characterised by spectroscopy. They were found to be

cytotoxic.

Lukianol A was synthesized using Diels Alder strategy. 183 Lukianol A was

convergently synthesized using Stille, Suzuki, or Negishi cross-coupling reactions as the

key step. 208 In 1999 Boger’s group reported lukianol A by utilizing a common

heterocyclic azadiene Diels-Alder reaction. 209 In 2000, Wong’s group reported the

formal total synthesis of lukianol A. 210 In 2006, Steglich et al. synthesized lukianol A in

which the lukianol skeleton is assembled in a single step. 211 In 2007, the same group

reported a short synthesis of lukianol A. 212

Dictyodendrins:

Dictyodendrins A (69), B (70), C (71), D (72) and E (73) were isolated from the

Japanese sponge Dictyodendrilla verongiformis. 213 They were found to possess

telomerase inhibitory activity.

42

Furstner et al. reported flexible total syntheses of the telomerase inhibitors

dictyodendrin B, C and E. 214

Sato et al. 215 reported three potent aldose reductase inhibitors (74, 75, 76) isolated

from a Japanese marine sponge, Dictyodendrilla species and were characterized by

chemical and physical evidence and X-Ray crystallographic analysis.

Aldose reductase catalyzes reduction of aldoses such as glucose and galactose, to

the corresponding polyols, such as sorbitol and galactol respectively. Intracellular

accumulation of the polyols may result in diabetic complications. Aldose reductase

inhibitors may therefore provide an effective means for the prevention and treatment of

such diseases.

Some other miscellaneous 3,4-diaryl pyrrole alkaloids are didemnimides,

granulatimides, rigidins, staurosporine aglycones, arcyriaflavins etc.

Didemnimides:

The alkaloids didemnimides A (77), B (78), C (79) and D (80) which inhibit

predation by fish, were isolated from the ascidian Didemnum conchyliatum, which was

collected from the sea grass blades in mangrove habitats in Bahamas. 216

Didemnimides A & B were synthesized by Hughes et al. 217 Didemnimide C was

synthesized in four steps by Steglich et al. 218 Piers et al also reported the synthesis of

didemnimide C in three steps. 219

Berlinck et al reported didemnimide E (81) from Didemnum granulatum. 220

43

Granulatimides:

Didemnum granulatum from Brazil contained the G 2 cell cycle checkpoint

inhibitors, granulatimide (82) and isogranulatimide 220 (83) the structures of which were

confirmed by synthesis. Isogranulatimide was isolated also from Didemnum

conchyliatum, from Bahamas. 221 Didemnum granulatum also yielded a novel alkaloid 6-

bromo granulatimide 222 (84).

Rigidins:

Rigidin A (85), a phosphodiesterase inhibitor, was isolated from the ascidian

Eudistoma rigida 223 and was synthesized by Sakamoto et al. 224 An additional synthesis of

rigidin was also reported. 225

Rigidins B (86), C (87), and D (88) were isolated from an Okinawan collection of

Cystodytes sp. 226 and these were found to be mildly cytotoxic towards L1210 murine

leukemia cell line.

Magedov et al. reported a four-step synthesis of alkaloids rigidins A, B, C and

D.227

Rigidin E (89) was isolated from a Papua New Guinea collection of a Eudistoma

species.228

Halitulin:

Halitulin (90) was isolated from the South African sponge Haliclona

tulearensis.229 Its structure is elucidated mainly on the basis of spectroscopic data as well

as chemical modifications. Halitulin was found to be cytotoxic against several tumor

44

cells, P-388 murine leukemia, A-549 human lung carcinoma, HT-29 human colon

carcinoma and MEL-28 human melanoma in concentrations of 12-25 ng/ml. It was

synthesized by Heinrich et al.230

Staurosporins and arcyriaflavins:

11-hydroxy staurosporine (91) was first isolated from the ascidian Eudistoma

species from Pohnpei231 and was found to be a cytotoxic protein kinase C inhibitor.

A West African Eudistoma species yielded arcyriaflavin A (92) and staurosporine

aglycone which was responsible for the strong cytotoxicity and protein kinase C activity

of crude extracts.232

The prosobranch mollusk Coriocella nigra contained an additional cytotoxic

staurosporin analogue 4-N-demethyl 11-hydroxy staurosporine (93) 233. Another

staurosporin derivative, 3’-demethoxy 3, 3’-dihydroxystaurosporine (94) along with 4-N-

demethyl 11-hydroxy staurosporine was isolated from Eudistoma toealensis and its

predatory flatworm Pseudoceros species. 234 Both the ascidian Eudistoma toealensis and

its predatory flatworm Pseudoceros species collected in Chuuk and Micronesia yielded

three new staurosporine235 derivatives (95, 96, and 97).

Laatch et al. 236 reported a new staurosporine, N-carboxamido-staurosporine (98)

isolated from the culture broth of the marine-derived Streptomyces sp. QD518 from the

Jiaozhou Bay of Quindao, China. The structures were determined by spectroscopic

methods and by comparison of the NMR data with those of structurally related known

natural products, which were isolated from the same strain.

45

Two new indolocarbazole alkaloids, 7-oxo-3, 8, 9-trihydroxystaurosporine (99)

and 7-oxo-8, 9-dihydroxy-4’-N-demethyl staurosporine (100), were isolated from the

samples of the marine ascidian Cystodytes solitus collected in Tanzania. Their structures

were determined by a combination of spectroscopic techniques. Both compounds

displayed strong cytotoxicity against three human cell lines. 237

3, 4-diaryl pyrrole alkaloids from marine organisms showed varied biological

activities and these are summarized in Table 1-03.

Table I-03. Biological activities of 3,4 diaryl pyrrole alkaloids from marine organisms.

Compound Activity Compound No.

Reference

lamellarin D Cytotoxic,

multi drug resistance reversal agent

Topoisomerase1 inhibitor Mitochondrial permeability-

transition inhibitor

4 [120,127,128]

[123]

[121,132,133] [136]

lamellarin K Immunomodulatory effects 11 [151]

lamellarin L 7-hydroxyneolamellarin A

Immunomodulatory effects Hypoxia-induciblefactor-1 inhibitor

12 22

[151] [166]

lamellarin α 20-sulfate HIV integrase inhibition 31 [172]

lamellarin β Cytotoxic 32 [174] lamellarin ζ Cytotoxic 35 [176]

lamellarin χ Cytotoxic 38 [176] lamellarin L- triacetate Cytotoxic 12a [176]

lamellarin F Cytotoxic 6 [176] ningalin B Multi drug resistance reversal 40 [184]

46

agent

storniamides A-D Antibiotic 43-46 [193] polycitone A Cell growth inhibitor

HIV-1RT inhibitor 47 [195]

[196] Purpurone

Baculiferins (C,E-H,K-N)

ATP-citrate lyase inhibitor

HIV-1 inhibitor 51

(54,56-59, 62-65)

[203]

[206]

lukianols A,B Cytotoxic 67,68 [207] dictyodendrins A-E Telomerase inhibitor 69-73 [213]

aldose-reductase inhibitors aldose-reductase inhibitors 74-76 [215] Granulatimide G2 cell cycle check point

inhibitor 82 [220]

Isogranulatimide G2 cell cycle check point inhibitor

83 [220]

rigidin A Phosphodiesterase inhibitor 85 [223]

rigidins B, C, D Cytotoxic 86-88 [226] Halitulin Cytotoxic 90 [229]

11-hydroxy staurosporine 7-oxo-3,8,9-trihydroxy

staurosporine

7-oxo-8,9-dihydroxy-4’-N-demethyl staurosporine

Cytotoxic protein kinase C inhibitor

Cytotoxic

Cytotoxic

91

(99)

(100)

[231]

[237]

[237]

47

LAMELLARINS

O

NX

HO

MeO

HO

MeOMeO OMe

OMe

O

(1) Lamellarin A X = OH(3) Lamellarin C X = H

O

N

HO

MeO

HOMeO OR

X

O

OMe(2) Lamellarin B R=Me X = OMe(4) Lamellarin D R=X = H

O

N

R1O

R2O

MeOMeO OR4

R5

O

OR3

(5) Lamellarin E R1=R3=H R2= R4=Me R5= OH (6) Lamellarin F R1= H R2=R3=R4=Me R5= OH(7) Lamellarin G R1= Me R2=R3=R4=R5=H Lamellarin G 8-sulfate R4=SO3

O

N

HO

HO

HOHO OH

O

OH

(8) Lamellarin H

O

N

HO

MeO

R2OMeO OR3

X

O

R1O

(09) Lamellarin I R1=R2=R3= Me X=OMe (10) Lamellarin J R1=R2= Me R3 =X=H (11) Lamellarin K R1=R3= Me R2=H X=OH (12) Lamellarin L R1=Me R2=R3=X=H

O

N

AcO

MeO

HOMeO OAc

O

AcO

(12a) Lamellarin L triacetate

48

O

N

SO3-O

MeO

HOMeO OH

O

MeO

(12b) 20 sulfate of Lamellarin L

O

N

HO

MeO

R2OMeO OR3

X

O

R1O

(13) Lamellarin M R1=R3= Me R2= H X=OH (14) Lamellarin N R2= Me R1= R3 =X=H

N COOMe

HO OH

OMe

OX

(15)Lamellarin O X=H(16)Lamellarin P X=OH (18) Lamellarin R

N COOMe

HO OH

OH

NH

COOMe

HO OH

(17)Lamellarin Q

O

N

HO

HO

HOMeO OH

O

OH

(23) Lamellarin S

49

NR O

HO OH

OH(19)Neolamellarin A R=H(22)7-Hydroxyneolamellarin A R=OH

NO

HO OH

OH(20) Neolamellarin B R=H(21) 5-Hydroxyneolamellarin B R=OH

R O

O

N

R1O

MeO

MeOR2O OMe

O

OH

(25) Lamellarin U R1=H R2=Me 20-Sulfate of lamellarin U R1=SO3Na(29) Lamellarin Y R1=SO3Na R2=H

O

N

HO

MeO

MeOMeO OR

O

OH

(27) Lamellarin W R=Me X=OMe (28) Lamellarin X R=Me X=OH

X

O

N

RO

MeO

MeOMeO OMe

O

OH(24) Lamellarin T R=X=H (26) Lamellarin V R=H X=OH 20-Sulfate of lamellarin T R=SO3Na20-Sulfate of lamellarin V R=SO3Na

X

OMe

O

N

MeO

HO

HOMeO OH

O

OH

(30) Lamellarin Z

50

O

N

NaO3SO

MeO

MeOMeO OMe

O

OH

O

N

HO

HO

MeOHO OH

O

OH

(31) Lamellarin α 20-sulfate (32) Lamellarin β

O

N

HO

MeO

MeO OMe

O

OMe

MeO OH

O

N

HO

MeO

MeO OMe

O

OMeMeO

(33) Lamellarin γ (34) Lamellarin ε

O

N

R1O

MeO

R2O OMe

O

OMeR3OX

O

N

AcO

MeO

MeO OAc

O

OMeAcOH

(35) Lamellarin ζ R1=H R2=R3=Me (38) Lamellarin χ

X=OMe

(36) Lamellarin η R1=H R2=R3=Me

X=H

(37) Lamellarin R1=R2=R3=Ac

X=OMe

51

NH

OO

O O

HO OH HOOH

(39) Ningalin A

NO

HO OH HO OH

HO OH(40) Ningalin B

N

HO OH HO OH

HO OH(41) Ningalin C

O

OHOH

O

N

HO OH

HO OH

(42) Ningalin D

OO

HO

HO

NHN

HOR2

HN

OHR3

OHHO

HO

OH OH

R1

OH

O O

Storniamides A R1=OH R2=R3=H(43) B R1=R3=OH R2=H(44) C R1=H R2=R3=OH(45) D R1= R2=R3=OH(46)

52

N

BrHO

Br

BrOH

Br

OH

O O

(47) Polycitone A

Br

OH

Br

BrHO

Br

NH

BrHO

Br

BrOH

Br

O O

(48) Polycitone B

BrOH

Br

BrHO

Br

N

BrHO

Br

BrOR

Br

(49) Polycitrin A R=H(50) Polycitrin B R=Me

OO

OH

N

HO OH HO OH

(51) Purpurone

O O

HO

HO

HO

OH

OH

N

HO OR1 R2O OH (52) Baculiferin A R1=SO3H R2=R3=R4=H(53) Baculiferin B R3=SO3H R1=R2=R4=H(54) Baculiferin C R3=R4=SO3 H R1=R2=H(55) Baculiferin D R1=R2=SO3H R3=R4=H(56) Baculiferin E R1=R3=SO3H R2=R4=H(57) Baculiferin F R1=R4=SO3H R2=R3=H(58) Baculiferin G R1=R3=R4=SO3H R2=H(59) Baculiferin H R1=R2=R3=SO3H R4=H

O O

R3O

HO

HO

OH

OR4

53

N O

HO OHOH

(60) Baculiferin I R1=SO3H R2=R3=H(61) Baculiferin J R1=H R2=SO3H R3=H(62) Baculiferin K R1=R2=34=H

O

HO

HO

OR2

R3

OR1

N

HO OR HOOH

(63) Baculiferin L R=H(64) Baculiferin M R=SO3H

O O

HO

HO OH

OHCOOH

N O

HO OHOH

(65) Baculiferin N

O

HO

HO

OH

COOH

NH

OO

HO OR HOOH

(66) Baculiferin O R=SO3H

O O

N

HO

X

OH

(67) Lukianol A X=H(68) Lukianol B X=I

O

OH

O

N

HO

(69) Dictyodendrin AOH

NH

OSO3Na

OH

OH

MeOOC

N

HO

(70) Dictyodendrin BOH

NH

OSO3Na

OH

OH

O

54

N

HO

(71) Dictyodendrin C R=H(72) Dictyodendrin D R=SO3Na

OR

NH

OSO3Na

O

OH

ON

HO

(73) Dictyodendrin EOH

NH

OSO3Na

O

OHOH

N

HO

(74) Aldose reductase inhibitor 1. M=Na(75) Aldose reductase inhibitor 2. M=H

OH

NH

OSO3M

O

OH

O

OH

HO

N

HO

(76) Aldose reductase inhibitor 3OH

NH

OSO3H

O

OHOH

MeOOC

NH

NNHN

R1

R2

O O

(77)Didemnimide A R1=R2=H (78) B R1=Br R2=H (79) C R1=H R2=Me (80) D R1=Br R2=Me (81) E R1=Me R2=H

NH

O O

(82) Granulatimide

HNN

HN

NH

O O

(83) Isogranulatimide

N

HNN

NH

O O

(84) 6-Bromogranulatimide

HNN

HN

Br

55

NH

N

NH

OHR2

O

O

O

R3

R1 OH

(85) Rigidins A R1=R2=R3=H (86) B R1=R3=H R2=OMe (87) C R1=OMe R2=R3=H (88) D R1=R2=OMe R3=H (89) E R1=R2=H R3=Me

N

HO OH

(90) Halitulin

N N

HO OH

N

NH

O

(91) 11-Hydroxystaurosporine

NNHO O

MeONHMe

NH

O

(92) Arcyriaflavin A

HN

HN

O

NH

O

(93)4'-N-demethyl11-hydroxystaurosporine

NNHO O

MeONH2

56

NH

O

(94) 3'-demethoxy-3,3'-dihydroxystaurosporine

NN O

HONHMe

OH

NH

O

(95) R1=H R2=R3=R4=Me R5=OH(96) R1=R3= R4=H R2=Me R5=OH(97) R1= R2 =R3= R4 = R5= H

NN O

R2ON

R5

R1

R4 R3

NH

O

(98) N-Carboxamido-staurosporine

NN O

NH3C CONH2

H3CO

NH

O

(99) 7-oxo-3,8,9-trihydroxystaurosporine (R1=OH, R2=Me )(100) 7-oxo-8,9-dihydroxy- 4'-N-demethylstaurosporine (R1=R2=H )

NN O

NHR2H3CO

O

OHHO R1

57

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